The present invention relates to compositions and methods for the prevention, prognostic evaluation, and treatment of oncogenic disorders, especially breast cancer, wherein a protein tyrosine kinase capable of complexing with a member of the SH2-and/or SH3-domain containing family of adaptor proteins is involved.
Specifically, the present invention relates to compositions and methods for decreasing or inhibiting the interaction between the components of protein tyrosine kinase/adaptor protein complexes, and/or decreasing or inhibiting the activity of such complexes, especially HER2/GRB-7 complexes, and to methods for the identifying such agents. Further, the present invention relates to the use of such methods and compositions for the treatment of the oncogenic disorders of interest, especially breast cancer. Still further, the present invention relates to compositions and methods for the treatment of oncogenic disorders, especially breast cancer, which involve modulating the activity and/or level of individual components of the protein tyrosine kinase/adaptor protein complexes, and additionally relates to methods for the identification of agents for such treatments.
Cells rely, to a great extent, on extracellular molecules as a means by which to receive stimuli from their immediate environment. These extracellular signals are essential for the correct regulation of such diverse cellular processes as differentiation, contractility, secretion, cell division, contact inhibition, and metabolism. The extracellular molecules, which can include, for example, hormones, growth factors, lymphokines, or neurotransmitters, act as ligands that bind specific cell surface receptors. The binding of these ligands to their receptors triggers a cascade of reactions that brings about both the amplification of the original stimulus and the coordinate regulation of the separate cellular processes mentioned above. In addition to normal cellular processes, receptors and their extracellular ligands may be involved in abnormal or potentially deleterious processes such as virus-receptor interaction, inflammation, and cellular transformation to a cancerous state.
A central feature of this process, referred to as signal transduction (for reviews, see Posada, J. and Cooper, J. A., 1992, Mol. Biol. Cell 3:583-592; Hardie, D. G., 1990, Symp. Soc. Exp. Biol. 44:241-255), is the reversible phosphorylation of certain proteins. The phosphorylation or dephosphorylation of amino acid residues triggers changes, such as in conformation, in regulated proteins that alter their biological properties. Proteins are phosphorylated by protein kinases and are dephosphorylated by protein phosphatases. Protein kinases and phosphatases are classified according to the amino acid residues they act on, with one class being serine-threonine kinases and phosphatases (reviewed in Scott, J. D. and Soderling, T. R., 1992, Current Opinion in Neurobiology 2:289-295), which act on serine and threonine residues, and the other class being the tyrosine kinases and phosphatases (reviewed in Fischer, E. H. et al., 1991, Science 253:401-406; Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212), which act on tyrosine residues. The protein kinases and phosphatases may be further defined as being receptors, i.e., the enzymes are an integral part of a transmembrane, ligand-binding molecule, or as non-receptors, meaning they respond to an extracellular molecule indirectly by being acted upon by a ligand-bound receptor. Phosphorylation is a dynamic process involving competing phosphorylation and dephosphorylation reactions, and the level of phosphorylation at any given instant reflects the relative activities, at that instant, of the protein kinases and phosphatases that catalyze these reactions.
While the majority of protein phosphorylation occurs at serine and threonine amino acid residues, phosphorylation at tyrosine residues also occurs, and has begun to attract a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression, metastasis, and neoplastic transformation is now well established (Cantley, L. C. et al., 1991, Cell 64:281-302; Hunter T., 1991, Cell 64:249-270; Nurse, 1990, Nature 344:503-508; Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212). Subversion of normal growth control pathways leading to oncogenesis has been shown to be caused by activation or overexpression of protein tyrosine kinases which constitute a large group of dominant oncogenic proteins (reviewed in Hunter, T., 1991, Cell 64:249-270).
Protein tyrosine kinases comprise a large family of proteins, including many growth factor receptors and potential oncogenes, which share ancestry with, but nonetheless differ from, serine/threonine-specific protein kinases (Hanks et al., 1988, Science 241:42-52).
Receptor-type protein tyrosine kinases having a transmembrane topology have been studied extensively. The binding of a specific ligand to the extracellular domain of a receptor protein tyrosine kinase is thought to induce receptor dimerization and phosphorylation of their own tyrosine residues. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signalling molecules, thereby activating various signal transduction pathways (Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212).
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases, may be broadly defined as those protein tyrosine kinases which do not contain a hydrophobic, transmembrance domain. Members of the various morphotypic families of cytoplasmic protein tyrosine kinases which have been identified share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains include the SH2 (SRC homology domain 2; Sadowski, I. et al., Mol. Cell. Biol. 6: 4396-4408; Koch, C. A. et al., 1991, Science 252:668-674) domains and SH3 domains (SRC homology domain 3; Mayer, B. J. et al., 1988, Nature 332:269-272). The non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction (Pawson, T. and Gish, G., 1992, Cell 71:359-362).
Intracellular proteins having characteristic conserved peptide domains (SH2 and/or SH3 domains, as described below) which are critical to the signal transduction pathway. Such proteins, which may be termed adaptor proteins, link protein tyrosine kinases, especially receptor-type protein tyrosine kinases to downstream intracellular signalling pathways such as the RAS signalling pathway. It is thought that such adaptor proteins may be involved in targeting signal transduction proteins to the correct site in the plasma membrane or subcellular compartments, and may also be involved in the regulation of protein movement within the cell.
Such adaptor proteins are among the protein substrates of the receptor-type protein tyrosine kinases, and have in common one or two copies of an approximately 100 amino acid long motif. Because this motif was originally identified in c-Src-like cytoplasmic, non-receptor tyrosine kinases it is referred to as a Src homology 2 (SH2) domain. SH2-containing polypeptides may otherwise, however, be structurally and functionally distinct from one another (Koch, C. A. et al., 1991, Science 252:668-674). SH2 domains directly recognize phosphorylated tyrosine amino acid residues. The peptide domains also have independent sites for the recognition of amino acid residues surrounding the phosphotyrosine residue(s).
When a receptor protein tyrosine kinase binds an extracellular ligand, receptor dimerization is induced, which, in turn, leads to intermolecular autophosphorylation of the dimerized kinases (Schlessinger, J. and Ullrich, A., 1992, Neuron 9: 383-391). Receptor phosphorylation, therefore, creates SH2-binding sites, to which an adaptor protein may bind.
In addition to SH2 peptide domains, many of the adaptor proteins involved in signal transduction contain a second conserved motif of 50-75 amino acids residues, the SH3 domain (Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Pawson, T. and Gish, G. D., 1992, Cell 72:359-362; Mayer, B. J. and Baltimore, D., 1993, Trends in Cell Biol. 3 8-13; Mayer, B. J. et al., 1988, Nature 352:272-275). Much less is known about the biological role of the SH3 domain than is known about the role of SH2. The current view is that SH3 domains function, in part, as protein-binding domains that act to link signals transmitted from the cell surface to downstream effector genes such as ras (Pawson, T. and Schlessinger, J., 1993 Current Biology, 3:434-442).
Growth factors and their receptors are crucial for normal development but can also act as oncogenes leading to cell transformation and cancer. Among women, breast cancer is by far the leading cause of cancer, with invasive breast cancer affecting approximately one woman in nine. (Lippman, M. E., 1993, Science 259:631-632).
A number of proteins have been identified which may participate in the aberrant growth of breast cancer cells. Such proteins include p53, a transcriptional regulator with tumor suppressor properties, nm23, a putative metastasis suppressor, and several families of cell surface growth factor receptors and their cognate ligands, including the epidermal growth factor (EGF) receptor superfamily, the insulin-like growth factor (IGF-1) family, and the fibroblast growth factor (FGF) family. For example, HER2, a receptor with close similarity to EGF-Receptor, also known as c-erBb-2 (Coussens et al., 1985, Science 230:1132-1139; Yamamoto et al., 1986, Nature 319:230-234; King et al., 1985, Nature 307:521-527) has been identified. This receptor was also isolated as the rat oncogene neu, an oncogene responsible for chemically induced rat glioblastomas (Padhy et al., 1982, Cell 28:865-871; Schechter et al., 1984, Nature 312:513-516; Bargmann et al., 1986, Nature 319:226-230). HER2/erbB-2 is known to be amplified and overexpressed in about 25% of human breast cancers (Slamon et al., 1987, Science 235:177-182; Slamon et al., 1989, Science 244:707-712).
To date, however, none of the identified proteins has yielded a successful clinical therapy. That is, while great progress has been made in detection and treatment of localized disease, there has been only relatively modest progress in the treatment of advanced disease. Clearly, new therapeutic approaches are needed.
The present invention relates to compositions and methods for the prevention, prognostic evaluation, and treatment of oncogenic disorders, especially breast cancer, wherein a protein tyrosine kinase capable of complexing with a member of the SH2-and/or SH3-containing family of adaptor proteins is involved.
Specifically, the present invention relates to compositions and methods for decreasing or inhibiting the interaction between the components of protein tyrosine kinase/adaptor protein complexes, and/or decreasing or inhibiting the activity of such complexes, especially HER2/GRB-7 complexes, and to methods for identifying such agents. Further, the present invention relates to the use of such methods and compositions for the treatment of the oncogenic disorders of interest, especially breast cancer. Still further, the present invention relates to compositions and methods for the treatment of oncogenic disorders, especially breast cancer, which involve modulating the activity and/or level of individual components of the protein tyrosine kinase/adaptor protein complexes, and relates to methods for the identification of agents for such treatments. Additionally, the present invention relates to methods and compositions for prognostic evaluation of oncogenic disorders, especially breast cancer, which involve protein tyrosine kinase capable of complexing with a member of the SH2- and/or SH3-containing family of adaptor proteins.
xe2x80x9cProtein tyrosine kinasexe2x80x9d will, herein, be abbreviated xe2x80x9cPTKxe2x80x9d. It is to be understood that xe2x80x9cPTKxe2x80x9d may refer to either a transmembrane, receptor-type protein tyrosine kinase or a cytoplasmic protein tyrosine kinase, unless otherwise indicated.
This invention is based, in part, on the discovery that an adaptor protein, GRB-7, is overexpressed in several different breast cancer cell lines and in tissue samples from primary human breast cancer, and, further, on the discovery that GRB-7 is amplified in concert with the receptor tyrosine kinase molecule HER2, a signal transduction molecule implicated in the development of breast cancer. The present invention is further based on the surprising discovery that GRB-7, via its SH2 domain, tightly binds HER2, and that, in fact, a large fraction of tyrosine phosphorylated HER2 in the breast cancer cell line SKBR-3 is bound to GRB-7. The data representing these discoveries is presented in the Working Examples in Sections 7 and below. Additional Working Examples demonstrate that GRB-7 maps to human chromosome region 17q near HER2 and the breast cancer susceptibility gene, BRCA1 (Section 6), GRB-7 forms a complex with the tyrosine phosphorylated protein SHC (Section 8), GRB-7 is phosphorylated by activated HER2 (Section 9), and it is shown that GRB-7 contains a pleckstrin domain, a domain that functions in binding to other regulatory factors (Section 10). The Example presented in Section 11 involves a screening assay for the identification of substances that inhibit the physical interaction between an adaptor protein (GRB-7 in this Example) and an activated tyrosine kinase molecule (HER2, in this Example).